GenWay Biotech, Inc. 6777 Nancy Ridge Drive San Diego, CA 92121 Phone: 858.458.0866 Fax: 858.458.0833 Email: techline@genwaybio.com http://www.genwaybio.com Human VEGF ELISA Kit Catalog Number: Size: GWB-ZZK029 96 wells/kit Sandwich High Sensitivity ELISA kit for Quantitative Detection of Human VEGF in cell culture supernates, serum, and plasma (heparin, EDTA, citrate). Typical Data Obtained from Human VEGF (TMB reaction incubate at 37 C for 20-25min) Concentration(pg/ml) 0 31.2 62.5 125 250 500 1000 2000 O.D. 0.024 0.110 0.175 0.312 0.573 0.976 1.597 2.140 Typical Human VEGF ELISA Kit Standard Curve This standard curve was generated for demonstration purpose only. A standard curve must be run with each assay. Range Sensitivity Specificity Cross-reactivity 31.2 pg/ml 2000 pg/ml (human serum, plasma) 15.6 pg/ml 1000 pg/ml (cell culture supernates) <1 pg/ml Natural and recombinant Human VEGF There is no detectable cross reactivity with other relevant proteins
Storage Store at 4 C for 6 months, at -20 C for 12 months. Avoid multiple freeze-thaw cycles (Shipped with wet ice.) Intra/Inter Assay Precision Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision. Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision. Intra-Assay Precision Inter-Assay Precision Sample 1 2 3 1 2 3 n 16 16 16 24 24 24 Mean(pg/ml) 146 650 1173 242 567 1084 Standard deviation 6.57 24.7 63.34 12.1 27.78 70.46 CV(%) 4.5 3.8 5.4 5 4.9 6.5 Assay Principle The Human VEGF ELISA Kit was based on standard sandwich enzyme-linked immune- sorbent assay technology. A monoclonal antibody specific for VEGF has been precoated onto 96-well plates. Standards(Expression system for standard: sf21; Immunogen sequence: A27-R191) and test samples are added to the wells, a biotinylated detection polyclonal antibody specific for VEGF is added subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex was added and unbound conjugates were washed away with PBS or TBS buffer. HRP substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the Human VEGF amount of sample captured in plate. Kit Components Materials included in the kit Description Quantity 96-well plate precoated with anti- human VEGF antibody 1 Lyophilized recombinant human VEGF standard 10ng/tubex2 Biotinylated anti- human VEGF antibody 130ul(dilution 1:100) Avidin-Biotin-Peroxidase Complex (ABC) 130ul(dilution 1:100) Sample diluent buffer 30ml Antibody diluent buffer ABC diluent buffer TMB color developing agent TMB stop solution Adhesive Cover 4 12ml 12ml 10ml 10ml
Materials Required But Not Provided 1. Microplate reader in standard size. 2. Automated plate washer. 3. Adjustable pipettes and pipette tips. Multichannel pipettes are recommended in the condition of large amount of samples in the detection. 4. Clean tubes and Eppendorf tubes. 5. Washing buffer (neutral PBS or TBS). Preparation of 0.01M TBS: Add 1.2g Tris, 8.5g NaCl; 450μl of purified acetic acid or 700μl of concentrated hydrochloric acid to 1000ml distilled water and adjust ph to 7.2~7.6. Finally, adjust the total volume to 1L. Preparation of 0.01 M PBS: Add 8.5g sodium chloride, 1.4g Na 2 HPO 4 and 0.2g NaH 2 PO 4 to 1000ml distilled water and adjust ph to 7.2~7.6. Finally, adjust the total volume to 1L. Notice Before Application Please read the following instructions before starting the experiment. 1. To inspect the validity of experiment operation and the appropriateness of sample dilution proportion, pilot experiment using standards and a small number of samples is recommended. 2. The TMB Color Developing agent is colorless and transparent before using, contact us freely if it is not the case. 3. Before using the Kit, spin tubes and bring down all components to the bottom of tubes. 4. Duplicate well assay is recommended for both standard and sample testing. 5. Don t let 96-well plate dry, for dry plate will inactivate active components on plate. 6. Don t reuse tips and tubes to avoid cross contamination. 7. Avoid using the reagents from different batches together. 8. In order to avoid marginal effect of plate incubation due to temperature difference (reaction may be stronger in the marginal wells), it is suggested that the diluted ABC and TMB solution will be pre-warmed in 37 for 30 min before using. 9. Take precautionary measures to prevent operator contamination (such as saliva and other body fluids) of kit reagents while running this assay. Preparation 1. Sample Preparation and Storage Store samples to be assayed within 24 hours at 2-8 C. For long-term storage, aliquot and freeze samples at -20 C. Avoid repeated freeze-thaw cycles. Serum: Allow the serum to clot in a serum separator tube (about 4 hours) at room temperature. Centrifuge at approximately 1000 X g for 15 min. Analyze the serum immediately or aliquot and store samples at -20 C. Plasma: Collect plasma using heparin, EDTA, or citrate as an anticoagulant. Centrifuge for 20 min at 2000 x g within 30 min of collection. Analyze immediately or aliquot and store fozen at -20 C. Cell culture supernates: Remove particulates by centrifugation, assay immediately or aliquot and store samples at -20 C. 2. Sample Dilution Guideline The user needs to estimate the concentration of the target protein in the sample and select
a proper dilution factor so that the diluted target protein concentration falls near the middle of the linear regime in the standard curve. Dilute the sample using the provided diluent buffer. The following is a guideline for sample dilution. Several trials may be necessary in practice. The sample must be well mixed with the diluents buffer. High target protein concentration (20-200ng/ml). The working dilution is 1:100. i.e. Add 1μl sample into 99 μl sample diluent buffer. Medium target protein concentration (2-20ng/ml). The working dilution is 1:10. i.e. Add 10μl sample into 90 μl sample diluent buffer. Low target protein concentration (31.2-2000pg/ml). The working dilution is 1:2. i.e. Add 50μl sample to 50 μl sample diluent buffer. Very Low target protein concentration (0-31.2pg/ml). No dilution necessary, or the working dilution is 1:2. 3. Reagent Preparation and Storage A. Reconstitution of the Human VEGF standard: VEGF standard solution should be prepared no more than 2 hours prior to the experiment. Two tubes of VEGF standard (10ng per tube) are included in each kit. Use one tube for each experiment. a. 10000pg/ml of Human VEGF standard solution: Add 1ml sample diluent buffer into one tube, keep the tube at room temperature for 10 min and mix thoroughly. b. 2000pg/ml of Human VEGF standard solution: Add 0.2 ml of the above VEGF standard solution into 0.8 ml sample diluent buffer and mix thoroughly. c. 1000pg/ml 31.2pg/ml of Human VEGF standard solutions: Label 6 Eppendorf tubes with 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml respectively. Aliquot 0.3ml of the sample diluent buffer into each tube. Add 0.3ml of the above 2000pg/ml VEF standard solution into 1st tube and mix. Transfer 0.3ml from 1st tube to 2nd tube and mix. Transfer 0.3ml from 2nd tube to 3rd tube and mix, and so on. Note: The standard solutions are best used within 2 hours. The 10ng/ml standard solution should be stored at 4 C for up to 12 hours, or at -20 C for up to 48 hours. Avoid repeated freeze-thaw cycles. B. Preparation of biotinylated anti-human VEGF antibody working solution: The solution should be prepared no more than 2 hours prior to the experiment. a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing 0.1-0.2 ml more than total volume) b. Biotinylated anti-human VEGF antibody should be diluted in 1:100 with the antibody diluent buffer and mixed thoroughly. (i.e. Add 1μl Biotinylated anti- Human VEGF antibody to 99μl antibody diluent buffer.) C. Preparation of Avidin-Biotin-Peroxidase Complex (ABC) working solution: The solution should be prepared no more than 1 hour prior to the experiment. a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing 0.1-0.2 ml more than total volume) b. Avidin- Biotin-Peroxidase Complex (ABC) should be diluted in 1:100 with the ABC dilution buffer and mixed thoroughly. (i.e. Add 1μl ABC to 99μl ABC diluent buffer.)
Assay Procedure The ABC working solution and TMB color developing agent must be kept warm at 37 C for 30 min before use. When diluting samples and reagents, they must be mixed completely and evenly. Standard VEGF detection curve should be prepared for each experiment. The user will decide sample dilution fold by crude estimation of VEGF amount in samples. 1. Aliquot 0.1ml per well of the 2000pg/ml, 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml Human VEGF standard solutions into the precoated 96-well plate. Add 0.1ml of the sample diluent buffer into the control well (Zero well). Add 0.1ml of each properly diluted sample of Human cell culture supernates serum, or plasma to each empty well. See Sample Dilution Guideline above for details. It is recommended that each Human VEGF standard solution and each sample be measured in duplicate. 2. Seal the plate with a new adhesive cover provided and incubate at 37 C for 90 min. 3. Remove the cover, discard plate content, and blot the plate onto paper towels or other absorbent material. Do NOT let the wells completely dry at any time. 4. Add 0.1ml of biotinylated anti-human VEGF antibody working solution into each well, seal the plate with a new adhesive cover provided and incubate at 37 C for 60 min. 5. Wash plate 3 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay in the wells for 1 min. Discard the washing buffer and blot the plate onto paper towels or other absorbent material. (Plate Washing Method: Discard the solution in the plate without touching the side walls. Blot the plate onto paper towels or other absorbent material. Soak each well with at least 0.3 ml PBS or TBS buffer for 1~2 minutes. Repeat this process two additional times for a total of THREE washes. Note: For automated washing, aspirate all wells and wash THREE times with PBS or TBS buffer, overfilling wells with PBS or TBS buffer. Blot the plate onto paper towels or other absorbent material.) 6. Add 0.1ml of prepared ABC working solution into each well, seal the plate with a new adhesive cover provided and incubate at 37 C for 30 min. 7. Wash plate 5 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay in the wells for 1-2 min. Discard the washing buffer and blot the plate onto paper towels or other absorbent material. (See Step 5 for plate washing method.) 8. Add 90μl of prepared TMB color developing agent into each well, seal the plate with a new adhesive cover and incubate at 37 C in dark for 20-25min (Note: For reference only, the optimal incubation time should be determined by end user. And the shades of blue can be seen in the wells with the four most concentrated Human Neurotrophin-3 standard solutions; the other wells show no obvious color). 9. Add 0.1ml of prepared TMB stop solution into each well. The color changes into yellow immediately. 10. Read the O.D. absorbance at 450nm in a microplate reader within 30 min after adding the stop solution. For calculation, (the relative O.D.450) = (the O.D.450 of each well) (the O.D.450 of Zero well). The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The Human Neurotrophin-3 concentration of the samples can be interpolated from the standard curve. Note: if the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution. Summary 1. Add samples and standards and incubate the plate at 37 C for 90 min. Do not wash. 2. Add biotinylated antibodies and incubate the plate at 37 C for 60 min. Wash plate 3 times with 0.01M TBS. 3. Add ABC working solution and incubate the plate at 37 C for 30 min. Wash plate 5 times with 0.01M TBS.
4. Add TMB color developing agent and incubate the plate at 37 C in dark for 20-25min. 5. Add TMB stop solution and read.